Abstract
Plasma membrane antigens are critical for the classification and diagnosis of chronic mature B-cell lymphocytic malignancies, illustrated by the extensive use of the Matutes score for CLL diagnosis. Nevertheless, new surface markers that could be detected using flow cytometry are still needed for the diagnostic and classification of a number of chronic lymphoid malignancies. In this aim, proteomic tools could be of great interest in order to detect such antigens, but analysis of plasma membrane proteins is limited by the difficulty of their purification and separation of membrane proteins with usual 2-dimensional electrophoresis have often been poor. For all these reasons, we proposed to take advantage of the physiological production of plasma membrane microvesicles in order to study plasma membrane proteins of malignant lymphoid cells. Indeed, when eukaryotic cells are submitted to stress conditions (including mitogenic activation or apoptosis), the constitutive asymmetry between the inner and outer leaflet of the plasma membrane is disrupted and fragments are released from the plasma membrane, constituting the so-called microparticles (MPs). Although MPs reflect at least in vitro the cell state, little is known on their protein composition. We describe here the first set of experiments aiming to characterize the MPs proteome. First, two ways of triggering MPs formation from a T-lymphocytic cell line were analyzed using a 1D-gel approach coupled with Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). 390 proteins were identified in MPs among which 34% were localized to the plasma membrane. Replicate experiments from independent samples identified only very few nucleus or organelles proteins, but revealed a broad representation of plasma membrane proteins including seventeen hematopoietic clusters of differentiation and numerous signal transduction proteins. This approach was then successfully applied to circulating cells of a small-lymphocytic B-cell lymphoma. 414 proteins were identified in MPs, including 118 membrane proteins. In addition to various CD antigens, annexins, immunoglobulin chains and HLA antigens, proteins involved in the B-cell receptor signal transduction were recovered, including tyrosine kinases LYN, SYK, BTK, CSK, but also phospho-inositide 3-kinase (PI3K) phospholipase Cγ (PLCγ 2), hemopoietic cell kinase (HCK) and JAK1.
Proteomic analysis of MPs may represent a new tool to study plasma membrane proteins, displaying the advantages of reproducibility, minimal organelle contamination and potentially applicable to most cell types. Extensive proteomic analysis of MPs may thus be a way to investigate differential expression of plasma membrane components and to identify new pathology markers that could help for diagnosis and classification.
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